Medicament for lct poisoning

ABSTRACT

The medicament for the prevention or the relief of poisoning by large clostridial cytotoxins (LCTs), in particular  Clostridium difficile  toxins A and B (TcdA and TcdB), is characterized by containing as active ingredient at least one effector, namely an inhibitor or activator of the autocatalytic protease activity of LCTs (large clostridial cytotoxins).

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 12/376,036,which is the U.S. national stage of International applicationPCT/DE2007/000957, filed May 26, 2007 designating the United States andclaiming priority to German applications. DE 102006036373.6, filed Aug.2, 2006 and DE 102007004938.4, filed Jan. 26, 2007.

FIELD OF THE INVENTION

The invention relates to a medicament for the prevention or the reliefof poisoning by large clostridial cytotoxins (LCTs), in particularClostridium difficile Toxins A and B (TcdA and TcdB), Clostridiumsordellii lethal Toxin (TcsL) and Clostridium novyi α-Toxin (Tcnα).

BACKGROUND OF THE INVENTION AND INTRODUCTION OF THE INVENTION

Clostridium difficile is a gram-positive, spore-forming germ, growingstrictly anaerobically, which was only identified at the end of the1970s as an etiological agent of antibiotic-associated diarrhoea andpseudomembranous colitis. Since the 1990s, C. difficile has beenregarded as the most significant hospital germ in developed countries.As a consequence of the continuously expanding use of broad spectrumantibiotics, the incidence of C. difficile infections is constantlyincreasing further especially in people treated as in-patients.

The exotoxins toxin A (TcdA) and toxin B (TcdB) produced by C. difficileare responsible for the C. difficile-associated diseases. Variousstrains exist with different virulence and toxin production.Approximately one quarter of all strains produces no toxins.Toxin-forming strains produce almost always both toxins. TcdA is anenterotoxin which through cytotoxic damage to the enterocytes increasesthe permeability of the intestinal mucosa and hence initiates diarrhoea.TcdB is a cytotoxin which disturbs the electrolyte transportation and isresponsible for loss of fluid and functional disturbances of theintestine. The toxins TcdA and TcdB belong to the group of so-calledlarge clostridial cytoxins (LCTs) and consist respectively of a peptidechain with three functional domains, namely the C-terminal domain, whichis responsible for binding the toxin to the host cell membrane, thehydrophobic middle domain, which is made (co)responsible for thetranslocation process through the cellular membranes, and the N-terminaldomain, which has a glycosyltransferase function and imparts the toxicactivity of the molecule.

The uptake process of the toxins in the host cell is in fact not yetfully explained, however it is considered a fact that the toxins, afterbinding to a host cell receptor, arrive into the host cell byendocytosis, and that for the development of their toxicity theN-terminal catalytic domain is split off and is conveyed into thecytosol of the host cell. There, the catalytic domain glycolizesspecifically GTPases of the Rho sub-family (Rho, Rac and Cdc42), whichin turn are involved in an abundance of signal transduction cascades,and in this way blocks the respective signal transduction processes,which finally leads to the disaggregation of the cytoskeleton and tocell death.

In the prior art, hitherto it was assumed that the splitting off of thecatalytic N-terminal domain of the toxin peptide chains of TcdA and TcdBand also other “large clostridial toxins” LCT being catalyzed by acellular protease (Rupnik et al., 2005 and Pfeiffer et al., 2003).Corresponding evidence was not, however, able to be provided.

In the course of the investigations which form the basis of the presentinvention, it was now surprisingly found, however, that the cleavage ofTcdA and TcdB is an autocatalytic process, which is initiated byinositol phosphate (IP), and that consequently the toxins of Clostridiumdifficile, in addition to their catalytic function ofglycosyltransferase also have the function of a protease forauto-cleavage or autocatalytic cleavage.

This protease function was identified as aspartate protease. Ascatalytic centre of the protease function, the protein region wasidentified which comprises the amino acid sequence of amino acidposition AS 1653 to AS 1678 of TcdB according to sequence No. P18177(SwissProt/TrEMBL). The motif DXG (Rao et al. 1998) characteristic foraspartate proteases lies at the amino acid position 1665.

As inositol phosphate binding site, the protein region was identifiedwhich comprises the amino acid sequence of amino acid position AS 1517to AS 2142 of the TcdB protein according to sequence No. P18177(SwissProt/TrEMBL). This amino acid sequence constitutes aninosin-5-monophosphate-dehydrogenase (IMPDH) motif, which is composed oftwo regions, namely AS 1517-AS 1593 and AS 1918-AS 2142, which areseparated by a 325 amino acid long protein section without sequencehomology.

For the treatment of patients with C. difficile infections, firstly theinitiating antibiotic is discontinued, in so far as this is possible.The further treatment takes place exclusively symptomatically. With along-lasting or serious etiopathology, and when a discontinuance of theinitiating antibiotic is not possible for other reasons, metrondiazol orvancomycin is administered for therapy.

The disadvantages of the current antimicrobial therapy are manifold. Itis critical here above all that a disease which was initiated as aresult of the treatment of a different infection situation withantibiotics can not be effectively healed with an antimicrobial therapy.The background to this is the fact that C. difficile only occursrelatively rarely in the gut of healthy people and can not stand up tothe normal intestinal flora. If the normal intestinal flora is destroyedby antiobiotic therapy, C. difficile can establish itself and caneffectively colonize the gut. The antibiotic therapy directed against C.difficile leads in turn to the destruction of the intestinal flora andthereby causatively also prevents the development of a healthyintestinal flora. This also explains the large number of remissionswhich are to be observed after completion of the antimicrobial therapy.An additional disadvantage of the current therapy is the increasingoccurrence of multiresistant C. difficile strains in recent times. Thethreat thereby is that the sole therapy hitherto for diseases induced byC. difficile also will become useless and the number of deaths as aresult of C. difficile diseases will increase. In addition to this isthe fact that the antibiotics necessary for the treatment of a C.difficile infection are very expensive and normally are only used injustified cases as reserve antibiotics.

SUMMARY OF THE INVENTION

There is therefore an urgent need for medicaments which are suited forthe specific combatting (prevention, elimination, relief) of C.difficile infections, without the risk existing of the development ofresistances in the clostridia or also other bacteria, and withoutdamaging the natural bacteria flora of the patient concerned—inparticular his intestinal flora.

An object of the present invention is the provision of such amedicament.

A solution to the said problem consists in the provision of a medicamentof the type mentioned in the introduction, which is distinguished inthat it contains as active ingredient at least one effector, namely aninhibitor or an activator of the autocatalytic protease activity of LCTs(large clostridial cytotoxins), in particular of the autocatalyticprotease activity of Clostridium difficile toxin A (TcdA) and/orClostridium difficile toxin B (TcdB) and/or Clostridium sordellii lethaltoxin (TcsL) and/or Clostridium novyi α-toxin (Tcnα).

Both activators and also inhibitors of the autocatalytic proteaseactivity of LCTs are designated below as effectors of the autocatalyticprotease activity of LCTs.

If the active ingredient or effector is an inhibitor, then its antitoxiceffect is based on the fact that it inhibits the protease activity ofthe intact toxin, in particular of the TcdB or TcdA or TcsL or Tcnα, andthereby prevents the splitting off of the cytotoxically effectivefragment with glucosyltransferase function (in the case of TcdB and TcdAthat is the 63 kDa fragment).

Suitable inhibitors are chemical substances which inhibit the proteaseactivity of the toxins.

The term “chemical substance” in the above and following explanationsdesignates both inorganic and also organic compounds, ions and peptidesor proteins.

Preferred inhibitors are those chemical substances which inhibit theprotease activity irreversibly. An example of this is the substance EPNP(1,2-epoxy-3-(p-nitrophenoxy)-propane). The substance reactsirreversibly with aspartate residues in the catalytic centre ofproteases and thus inhibits the proteolytic effect.

Further protease inhibitors are known to the specialist in the art orcan be easily identified by him by known methods (computer modelling,high throughput screening). For example, to carry out a high throughputscreening, a peptide can be synthesized, the amino acid sequence ofwhich corresponds to the sequence of the protease cleavage site of theLCTs. By coupling this peptide for example with the dye AMC(7-amino-4-methyl-cumarin) by methods which are known to the specialistin the art, a probe can be generated. To carry out the high throughputscreenings, the labelled probe is then brought together with the toxinand the candidate substances. If the probe is split, then changes occurin the fluorescence spectrum. These changes are easy to detect bymethods with which the specialist in the art is familiar (fluorescencedetectors). Batches in which no change to the fluorescence spectrum areto be observed then contain potential protease inhibitors.

By way of example, a further method is described for the identificationof substances which influence the activation of the autocatalyticprotease activity of the PCTs. For this, the holotoxin or also suitabletoxin fragments can be used, which for example are coupled with a dye,the fluorescence of which is quenched in the non-split toxin or toxinfragment. Through the autocatalytic cleavage of the toxin or of thetoxin fragments, the quenching effect is removed. The changes in thefluorescence spectrum can be easily detected, as described.

Particularly suitable inhibitors, i.e. effectors with inhibitor functionare chemical substances, in particular proteins and, amongst theseespecially antibodies, which inhibit the autocatalytic protease activityof the LCTs by interacting with the active centre of the protease.

The term “interact” in the present context means any kind of reciprocalaction between the LCTs and a chemical substance, in particular aprotein, and comprises in particular covalent bonds such as for exampledisulphide bonds and non-covalent bonds, such as for example van derWaals forces, hydrophobic or electrostatic reciprocal actions andhydrogen bridge bonds.

Proteins are preferred here, and amongst these especially antibodieswhich interact with the TcdB protein region of AS 1500 to AS 1800, inparticular from AS 1653 to AS 1678 and especially with the DXG motif atposition 1665—respectively according to TcdB amino acid sequence No.P18177 (SwissProt/TrEMBL) or the protein regions equivalent orhomologous thereto of TcfA or TcsL or Tcnα. These equivalent/homologousprotein regions are, in the case of TcdA, the amino acid sequencesection of AS 1651 to AS 1675 according to TcdA amino acid sequence No.P16154 (SwissProt/TrEMBL) with the DXG motif at amino acid position AS1662, in the case of TcsL, the amino acid sequence section of AS 1654 toAS 1679 according to TcsL amino acid sequence No. Q46342(SwissProt/TrEMBL) with the DXG motif at amino acid position AS 1666,and in the case of Tcnα, the amino acid sequence section of AS 1641 toAS 1665 according to Tcnα amino acid sequence No. Q46149(SwissProt/TrEMBL).

Further suitable inhibitors (effectors with inhibitor functions) arechemical substances, in particular proteins and, amongst theseespecially antibodies, which inhibit the autocatalytic protease activityof the LCTs, by inhibiting the interaction of the inositol phosphatewith the toxin. By the IP bond being prevented, the proteolytic cleavageof the toxins does not occur.

Proteins are preferred here, and amongst these especially antibodieswhich interact with the TcdB protein regions of AS 1400 to AS 2300, inparticular of AS 1517 to AS 2142 and especially of AS 1517 to AS 1593 orAS 1918 to AS 2142—respectively according to TcdB amino acid sequenceNo. P18177 (SwissProt/TrEMBL)—or with the protein regions, equivalent orhomologous hereto, of the toxins TcdA or TcsL or Tcnα. Equally well,antibodies or other proteins can also be generated, which do notinteract directly with the inositol phosphate binding site, inparticular with the above-mentioned protein regions, but rather aredirected towards adjacent regions and hinder the IP binding stericallyand hence prevent the proteolytic cleavage of the toxins. Furthermore,antibodies or other proteins can be generated, which do not interactdirectly with the DXG motif of the protease function of the LCTs, butrather prevent the proteolytic cleavage by binding in adjacent proteinsections.

Suitable inhibitors (effectors with inhibitor function) are constitutedin addition by structural analogues of inositol phosphate (IP) and inparticular of inositol hexaphosphate (IP6), which instead of IP and inparticular of IP6 can occupy the reaction binding sites of LCT, inparticular TcdA and/or TcdB, and/or TcsL and/or Tcnα, but do not havethe initiator function of IP or IP6. These structural analogues aretherefore antagonists to the agonists IP (in particular IP6) and bringabout a competitive inhibition of the protease activity of LCT, inparticular of TcdA and/or TcdB and/or TcsL and/or Tcnα. Suitablestructural analogues are known to the specialist in the art or can beeasily identified by known test methods with which the specialist in theart is familiar (examples of these have already been described above).

Suitable inhibitors are, in addition, inhibiting substances for/ofinositol phosphate (synonyms: inositol phosphate inhibiting substance orinositol phosphate inhibitor), i.e. those inhibiting substances whichbind or modify inositol phosphate and in particular inositolhexaphosphate such that its capability of initiating the proteaseactivity of LCT, in particular of TcdA and/or TcdB and/or TcsL and/orTcnα is prevented. An example of such substances are bivalent ions suchas Ca²⁺, which enter into insoluble complexes with IP6. Similarsubstances are known to the specialist in the art or can be easilyidentified by test methods which are known and with which the specialistin the art is familiar (examples of these are already described above).

Further suitable inhibitors (effectors with inhibitor function) arechemical substances which suppress the formation of inositol phosphatesin the gut lumen of the patients (mammals, especially humans) or in thebody cells of the patients (mammals, especially humans) or destroyalready present inositol phosphate and thus prevent the proteolyticcleavage of the LCTs on penetration into the cytoplasm. Preferredexamples of such substances are lithium, VPA (valproic acid) or CBZ(carbamazepine).

Suitable activators, i.e. effectors with activator function, arechemical substances which activate the protease activity of the toxins.

The antitoxic effect of an activator is based on the fact that itinitiates the protease activity of the intact toxin, in particular ofthe TcdB or TcdA or TcsL or Tcnα, still before the toxin has bonded tothe host cell such that the split off fragment could arrive into thecell interior (cytosol). The activator consequently brings about asplitting off of the cytotoxically effective fragment with glucosyltransferase function (in the case of TcdB and TcdA, that is the 63 kDafragment) outside the host cell. The cytotoxically effective fragmentcan then no longer arrive into the cell interior and develop itscytotoxic effect there.

A particularly suitable activator (effector with activator function) isisolated (in contrast to cytosolic) inositol phosphate (IP), preferablyinositol hexaphosphate (IP6).

A medicament with this active ingredient has the advantage that LCTpresent in the patient's gut, in particular TcdB and/or TcdA and/or TcsLand/or Tcnα is already caused to cleave in the gut through the IPsupplied as medicament, i.e. before it can bind to gut cells or otherbody cells and act toxically.

Equally suited as activator (effector with activator function) is asubstance which, in an analogous manner to IP6, promotes theautocatalytic protease activity of LCT, in particular of TcdA and/orTcdB and/or TcsL and/or Tcnα.

Such substances are known to the specialist in the art or can be easilyidentified by known methods. In addition, modified variants of the “highthroughput assays” described above are also suitable for this. Here, thetoxin and the potential activator substances are added together and inthe course of time, changes in the fluorescence spectrum are sought.Batches in which intensive changes occur in a short period of timecontain suitable activators (effectors with activator function).

The catalytic centres of the protease function of the LCTs can also beused according to the invention as systematically administered antigens(vaccination substances) to produce an immunisation against the toxins.The subject of the present invention is therefore also a medicament forthe prevention or the relief of poisoning by LCT (=large clostridialcytotoxins), which is characterized in that it is suitable foradministration as a vaccine, and that it has the amino acid sequence ofthe catalytic centre of TcdB and/or TcdA and/or TcsL and/or Tcnα, whollyor fragments thereof as antigen active ingredient(s). The antigen activeingredient(s) is/are preferably selected from the following group ofprotein fragments:

-   -   DXG motif at position 1665 of the TcdB amino acid sequence No.        P18177 (SwissProt/TrEMBL),    -   the amino acid positions AS 1653 to AS 1678 of the TcdB amino        acid sequence No. P18177 (SwissProt/TrEMBL),    -   the amino acid positions AS 1500 to AS 1800 of the TcdB amino        acid sequence No. P18177 (SwissProt/TrEMBL),    -   the DXG motif at position 1662 of the TcdA amino acid sequence        No. P16154 (SwissProt/TrEMBL),    -   the amino acid positions AS 1651 to AS 1675 of the TcdA amino        acid sequence No. P16154 (SwissProt/TrEMBL)    -   the DXG motif at position 1666 of the TcsL amino acid sequence        No. Q46342 (SwissProt/TrEMBL)    -   the amino acid positions AS 1654 bis AS 1679 of the TcsL amino        acid sequence No. Q46342 (SwissProt/TrEMBL),    -   the amino acid positions AS 1641 to AS 1665 of the Tcnα amino        acid sequence No. Q46149 (SwissProt/TrEMBL).

BRIEF DESCRIPTION OF THE FIGURES

The invention is described in further detail below with the aid ofexample embodiments and figures, showing:

FIG. 1: Cleavage of the TcdB₁₀₄₆₃ (270 kDa) holotoxin into thetranslocation/ligand domain (207 kDa) and the N-terminal catalyticdomain (63 kDa) in SDS-PAGE, carried out with

-   -   a: a mixture of Cy3-marked TcdB₁₀₄₆₃ and pig spleen cell extract        (Example 1A);    -   b: a mixture of Cy3-marked TcdB₁₀₄₆₃ and pig spleen cell extract        freed of protein (Example 1B);    -   c: a mixture of Cy3-marked TcdB₁₀₄₆₃, and inositol phosphate;    -   d: a mixture of unmarked TcdB₁₀₄₆₃ and inositol phosphate;    -   e: a mixture of unmarked (purified by means of affinity        chromatography) TcdB₁₀₄₆₃ and inositol phosphate;    -   a-e respectively following an incubation at room temperature for        1 hour

FIG. 2: SDS-PAGE of a mixture of TcdB₁₀₄₆₃ and/or IP6, with or withoutpre-treatment of the toxin with EPNP;

-   -   Line 1: TcdB₁₀₄₆₃ after pre-treatment with EPNP and without IP6,        no band able to be evidenced in the 63 kDa range;    -   Line 2: TcdB₁₀₄₆₃ after pre-treatment with EPNP and after        incubation with IP6, the typical 63 kD band is only weakly        formed;    -   Line 3: TcdB₁₀₄₆₃ without pre-treatment with EPNP and after        incubation with IP6, a distinctly formed band can be seen in the        molecular weight range of 63 kDa;    -   Line 4: TcdB₁₀₄₆₃ without pre-treatment with EPNP and without        IP6, no band is able to be evidenced in the 63 kDa range;

FIG. 3: ESI-LCMSMS analysis after tryptic digestion of the native (a)and of the EPNP-modified (b) TcdB protein. MS survey scanes (largeimage) and fragmentation spectra (insert).

DESCRIPTION OF VARIOUS AND PREFERRED EMBODIMENTS

All the methods named in the following examples are known to thespecialist in the art and are described for example in Ausubel et al.(2003).

Example 1 Evidence of the Autocatalytic Protease Activity of TcdB

Clostridium difficile toxin B (270 kDa) of the reference strainVPI10463, abbreviated below to TcdB₁₀₄₆₃, was initiallyfluoresence-marked with Cy3.

For this, 200-400 μg TcdB₁₀₄₆₃ (tgcBIOMICS, Mainz, Germany) were markedwith the dye Cy3 in accordance with the instructions of the manufacturer(Amersham Biosciences), by the toxin being incubated with the dye,dissolved in diemethyl formamide, for 1 hour at 4° C. Non-bonded dye wasthen removed by means of size exclusion chromatography (═SEC), in which10 mM Tris-HCl pH 8.5 served as run buffer. The molar ratio between dyeand Cy3-marked TcdB₁₀₄₆₃ was 0.8-1.6. Marked TcdB₁₀₄₆₃ was aliquoted andstored at −80° C. until further use.

(A) To carry out the “In-vitro-cleavage-assay” known from the prior art(see in this respect in particular Rupnik et al (2005); reference ishereby made expressly to the content of this publication) an aliquotCy3-marked TcdB₁₀₄₆₃, thawed to room temperature, was incubated for 1hour at room temperature with pig spleen cell extract.

This pig spleen cell extract was produced as follows: Freshly obtainedpig spleen was held in phosphate buffer (PBS) and comminuted to a singlecell suspension. Through the addition of low salt buffer,—namely 150 mMNH₄Cl, 1 mM KHCO₃, 0.1 mM EDTA, pH 7.6-, erythrocytes which were presentwere lysed and thereby removed. Then the spleen cells were washed twicewith 10 mM Tris-HCl pH 8.5 and deep-frozen immediately at −80° C. Forthe desired cell extract, as required an aliquot of these deep-frozenspleen cells was thawed in an aliquot 10 mM Tris-HCl pH 8.5 andsuspended and this suspension was then subjected to an ultrasonictreatment. The obtained lysate was centrifuged for 1 hour at 200 000×gand 4° C. and the supernate was used for the upcoming experiments.

At the end of the incubation phase, the mixture of Cy3-marked TcdB₁₀₄₆₃and pig spleen extract was subjected to a SDS-PAGE, in order to separateand detect the TcdB fragments which occurred during the incubation. Theresult of this SDS-PAGE is illustrated in FIG. 1 a: As expected, the twoknown and characteristic 63 kDa and 207 kDa fragments of the Clostridiumdifficile Toxin B—here the TcdB₁₀₄₆₃—were obtained.

Aliquots of TcdB₁₀₄₆₃ without admixture of spleen cell extract served asnegative controls (see FIG. 1 “−”).

(B) In a parallel experiment, the spleen cell extract described in (A)was purified of protein before the incubation with the Cy3-markedTcdB₁₀₄₆₃. For this purpose, an aliquot of the pig spleen extractproduced according to (A) was subjected to six phenol-chloroformextractions, following the ultrasonic treatment, which extractions werecarried out as follows: Pig spleen extract was mixed withphenol-chloroform-isoamyl alcohol (25/24/1) in the volume ratio 1:1.This mixture was centrifuged for 10 minutes at 1,7000×g and 4° C. Theaqueous uppermost layer was decanted into a fresh centrifuge vessel andcentrifuging and decanting was repeated for a further five times. Inorder to also remove final residues of phenol, finally a chloroformextraction was carried out.

Aliquots of the aqueous and protein-free fraction of the spleen cellextract obtained in this way were diluted in the volume ratio 1:30 (30),1:100 (100) or 1:300 (300) with 10 mM tris-HCL pH8.5 and, as describedunder (A), mixed with aliquots of Cy3-marked TcdB₁₀₄₆₃, thawed to roomtemperature, and incubated for 1 hour at room temperature. At the end ofthe incubation phase, these mixtures were subjected to a SDS-PAGE, inorder to separate and detect the TcdB fragments which occurred duringthe incubation. The result of this SDS-PAGE was made visible by means ofthe Gel Doc EQ system image readers (BIO-RAD Munich, Germany) and isillustrated in FIG. 1 b: In all test batches the two characteristic 63kDa and 207 kDa fragments of TcdB₁₀₄₆₃ were obtained. This shows thatthe aqueous and protein-free fraction of the spleen cell extract stillhas or had the characteristic of splitting TcdB₁₀₄₆₃ into its twocharacteristic partial fragments.

In a further parallel experiment, the spleen cell extract described in(A) was treated with heat (96° C., 30 minutes), before it was incubatedas described in (A) with Cy3-marked TcdB₁₀₄₆₃ and was subjected to theSDS-PAGE. The result of this SDS-PAGE is likewise illustrated in FIG. 1b. Again, the two characteristic 63 kDa and 207 kDa fragments ofTcdB₁₀₄₆₃ were obtained.

The result shows that the heat-induced spleen cell extract continues tohave the characteristic of splitting TcdB₁₀₄₆₃ into its characteristicpartial fragments.

(C) In a further series of experiments, Cy3-marked TedB₁₀₄₆₃ andunmarked TcdB₁₀₄₆₃ were incubated alone (i.e. without the admixture ofspleen cell extract) with various inositol phosphates for 1 hour at roomtemperature and the respective mixtures were then subjected to aSDS-PAGE. The result of these investigations is illustrated in Table 1and in FIGS. 1 c-e and provide the surprising core conclusions, that theproteolytic cleavage of TcdB₁₀₄₆₃ is able to be initiated solely througha chemical substance such as IP, and that this proteolytic cleavage isconsequently an autocatalytic process of the toxin protein.

From Table 1, it can be seen that a range of inositol phosphates caninitiate the autocatalytic cleavage of TcdB₁₀₄₆₃. Inositol hexaphosphate(IP₆) brings about the strongest cleavage activity amongst the testedinositol phosphates, and this means W₆ has the highest initiatoractivity (see also FIG. 1 c-e).

Structural analogues or other substances related to the inositolphosphates, which have an initiator activity, are known to thespecialist in the art or can be easily determined by known methods (e.g.computer modelling). For example, the “High Throughput Assays” describedabove can also be used.

The testing out of different concentrations of IP₆ in the incubationexperiment with TcdB₁₀₄₆₃ shows (see FIGS. 1 c and 1 d) that for thecleavage of fluorescence-marked toxin B, an IP concentration of 10 μM(FIG. 1 c) is sufficient, whereas for the cleavage of unmarked toxin B(and only made visible in the SDS-PAGE by zinc stain (Zinc Stain andDestain Kit, Biorad, Hercules, USA)), still lower concentrations of upto 1 μm (FIG. 1 d) are sufficient.

Analogous experiments were also carried out with the LCTs TcdA₁₀₄₆₃,TcsL of C. sordellii and Tcnα. Here it was found that inositolphosphates have an activating effect on the autocatalytic cleavage ofall investigated toxins of the LCT family.

(D) In order to rule out that the TcdB₁₀₄₆₃, purified from culturesupernate of C. difficile, used in the experiments, was contaminatedwith proteases, a control experiment with especially purified TcdB₁₀₄₆₃was carried out. This purification of the TcdB₁₀₄₆₃ took place by meansof affinity chromatography with the use of the monoclonal antibody 2CV(DSM ACC 2321) as follows:

7 mg of the TcdB specific monoclonal antibody 2CV (ProteinG-purifiedsupernate of a serum-free hybridoma culture) was coupled to a HiTrap NHSsepharose column (commercially available at GE Healthcare, Freiburg,Federal Republic of Germany). Coupling and elution were carried outaccording to the instructions of the manufacturer. The finished columnwas charged with approximately 4 mg TcbB₁₀₄₆₃ and non-bonded proteinswere removed by washing three times with 50 mM tris/HCl, pH 7.0; 125 mMNaCl. The elution took place in one step with 0.1 M triethanolamine-HClpH11. The toxin eluted in three 4 ml fractions with concentrationsbetween 450-185 μg/ml. The eluted toxin was immediately neutralized with1M tris-HCl pH 7.5 in the volume ratio 1/10, which was guaranteed inthat these neutralizing solution had already been provided before thestart of the elution in the collecting tubes for the fractions. Theabsence of contaminating proteins was then demonstrated by SDS-PAGE andsubsequent zinc stain (see FIG. 1 e “−”).

The control experiment consisted of the incubation of unmarkedTcdB₁₀₄₆₃, purified in such a way, with IP₆, and subsequent SDS-PAGE anddemonstration of the toxin or of the toxin fragments by means of zincstain. The result of this experiment is illustrated in FIG. 1 e andshows the complete cleavage of the holotoxin into the two knownfragments 63 kDa and 207 kDa.

Example 2 Inactivation of TcdB-10463 Through Incubation with a ProteaseInhibitor

TcdB₁₀₄₆₃ was purified as described in Example 1 (D) by means ofaffinity chromatography with the use of the monoclonal antibody 2CV andthen pre-treated either (i) with the protease inhibitor EPNP (10 mM1,2-epoxy-3-(p-nitrophenoxy)-propane) or (ii)—as control—with buffer (50mM HEPES, 1M NaCl, 1 mM EDTA, pH 8.0) for 60 minutes at roomtemperature.

Then an in vitro cleavage assay analogous to Example 1 (A) was carriedout. The test batches comprised respectively a volume of 10 μl, andcontained respectively 50-100 ng unmarked TcdB₁₀₄₆₃, 100 μM IP6 and 10mM tris-HCl pH 8.5. Following the incubation (1 h at room temperature),these test batches were subjected to a SDS-PAGE (10%) and the toxins andtoxin fragments were then made visible by means of zinc stain.

FIG. 2 shows the result of this experiment: The incubation of TcdB₁₀₄₆₃alone with IP6 (Line 3) shows a distinctly marked band in the molecularweight range of 63 kDa. When the toxin is previously pre-treated withEPNP (Line 2), the typical 63 kD band is only weakly marked. Thisfinding shows that through the addition of EPNP, the proteolyticactivity (protease activity) of the TcdB₁₀₄₆₃ is almost completelyinhibited.

The toxin pre-treated with EPNP was additionally investigated in the CHOtest according to Moos et al. (2000) for its residual activity(cytotoxic effect).

The CHO test was carried out as follows: In a 96 well microtiter plate,CHO cells (=Chinese hamster ovarial cells) were disseminated (5000cells/well) and incubated for 16 hours under standard conditions (5%CO₂, DMEM F12 supplemented with 2 mN L-glutamins, 5% FCS). The toxinswere then introduced to the cells after gradual dilution in growthmedium. Dilution stages between 10° and 10⁻⁸ were tested. The cells wereincubated for 3 hours under standard conditions. Then the proportion ofrounded cells was determined microscopically, by several representativesections of the well being photographed and the elongated and therounded cells being counted. (See also Moos et al., Meth Enzymol. 2000,325: 114-125. Reference is made here expressly to the content of thispublication).

The results of this CHO test show that the TcdB₁₀₄₆₃ pre-treated withEPNP has a substantially weaker cytotoxic effect than the untreatedTcdB₁₀₄₆₃ (cf. Table 2). As the inhibiting effect of EPNP, as is known,is based on the fact that EPNP enters into covalent interactions withcatalytic aspartate residues and thereby brings about an irreversibleinactivation of the protease (Salto et al. 1994), the results of thepresent experiment show that the inhibiting of the TcdB₁₀₄₆₃ activity isbased on the inhibiting of a protease activity of this toxin molecule.

The experiment with EPNP described here proves that the toxic effect ofTcdB-₀₄₆₃ and other LCTs is significantly reduced by pre-treatment ofthe toxins with a suitable protease inhibitor.

EPNP constitutes a model substance for a covalent inhibitor of the LCTs.Further comparably covalently-acting or competitively-inhibitinginhibitors are known to the specialist in the art or can be determinedby him by known methods, for example by the already described “highthroughput assays”.

Example 3 Inactivation of the Cytotoxic Effect of TcdB₁₀₄₆₃ ThroughExtracellular Activation of the Protease Activity with IP6

TcdB₁₀₄₆₃ was incubated as described in Example 1 (C) with 100 μM IP6.Following the incubation, the smaller 63 kD fragment of toxin protein,split off by the protease activity, was separated off, by the batchbeing purified via microcon tubes (Millipore, exclusion size 100 kD).This 63 kDa fragment of TcdB₁₀₄₆₃ was then examined for rounding of thecells in the CHO test according to Moos et al. (2000) described inExample 2. Here, the protein was added undiluted and in dilution stagesto the cells.

In this experiment, it was found that the 63 kDa fragment, which has theglucosyltransferase function of Tcd₁₀₄₆₃, under the given conditionsalone is/was not able to bring about a cytotoxic effect. Neither indiluted nor in undiluted state a rounding of the cells (as a consequenceof a glucosilation of specific GTpases of the Rho sub-family, whichresults in a blocking of signal transduction processes which results ina disaggregation of the cytoskeleton) could be observed. This findingthat the toxin fragment generated by means of autocatalysis is inactiveextracellularly, confirms the results of Pfeifer et al. (2003) andRupnik et al (2005), which show that the split-off catalytic domain ofTcdB₁₀₄₆₃ is not taken up into eucaryontic cells and is thereforeinactive in the cell medium. (Whereas in these papers, however, anautocatalytic activity of the LCTs is explicitly ruled out [Pfeifer etal., 2003] or there is speculation concerning a cellular protease forthe activation of the LCTs [Rupnik et al., 2005], the experiment resultsobtained in connection with the present invention show for the firsttime that the N-terminal toxin fragment is split off autocatalytically).

The cytotoxic effect of TcdB₁₀₄₆₃ and other LCTs can consequently beinhibited in that the proteolytic cleavage of the toxins is alreadyinduced before penetration of the toxins into the cells.

Example 4 Demonstration of the Active Centre of the Protease of TcdB

EPNP-inactivated (cf. Example 2) and untreated TcdB10463 were separatedby means of SDS gel and represented by zinc stain. Then the bandscorresponding to the proteins were cut out and divided into smallpieces. These were removed of colour and dried, then reduced in 2 mM DTTand alkylated with 20 mM iodacetamide. After the washing and reneweddrying of the gel fragments, these were digested with trypsin overnightat 37° C. The resulting peptides were then separated by HPLC(NanoAcquity ultraperformance liquid chromatography, Waters, Milford,USA). For this, 4.1.1 of the samples were applied onto a reverse-phasecolumn (NanoEase BEH C₁₈ (75 μm×10 cm) of Waters, Milford, USA) in 2%mobile phase B buffer (0.1% formic acid in acetonitrile). Mobile phase Abuffer contained 0.1% formic acid in H₂O. Then the fragments were elutedthrough a gradient of 3-40% mobile phase B buffer (90 min at 300 nl/min)from the column.

The eluted fragments were then examined by mass spectrometry. For this,a Q-T of Premier mass spectrometer of the company Waters was used. Theapparatus was calibrated with a [Glu-1]-fibrinogen peptide solution (500fmol/μl at 300 nl/min) via the reference-sprayer of the NanoLockSpraysource (Waters). For the analysis of the results, the MassLnyx4.1software (Waters) was used.

The analysis of the results shows that the untreated TcdB differs fromthe EPNP-treated toxin only in a tryptic fragment (Illustration 3). Thisfragment comprises the amino acids AS 1653 to 1678 of the TcdB proteinaccording to TcdB amino acid sequence No. P18177 (SwissProt/TrEMBL) withthe DXG motif at Position 1665, characteristic for aspartate proteases.

A comparison of the amino acid sequence AS 1653 to AS 1678 of thecatalytic centre of TcdB with the corresponding catalytic centres andprotein regions of the toxins TcdA, TcsL and Tcnα shows that the regionis highly conserved (see Tab. 3).

The specialist in the art can therefore generate antibodies or otherproteins by known methods, which interact specifically with the activecentre of the protease domain of the toxins, and therefore for examplecan prevent the autocatalytic cleavage of the LCTs. Equally well,antibodies or proteins can be generated, which do not directly block theactive centre, but rather are directed to adjacent regions and henceprevent the autoproteolytic cleavage of the toxin sterically.

Example 5 Inhibition of the TcdB Effect Through Antibodies as a Resultof Immunisation with Inactivated TcdB

In order to induce a protection against the cytotoxic effects of TcdB,the following TcdB preparations were produced and used for immunisationin rabbits:

-   -   Preparation A: TcdB, inactivated with formalin    -   Preparation B: TcdB, inactivated with EPNP (cf. Example 2)    -   Preparation C: TcdB fragment AS1601-1716 (DSG motif)    -   Preparation D: TcdB fragment AS1508-1601 (part of the        inosine-binding motif)    -   Preparation E: TcdB fragments AS 1508-2157        -   (DSG motif and entire inosine-binding motif)

The TcdB fragments were expressed by methods known to the specialist inthe art in the plasmid pET-19 (Novagen) and purified via the attachedHis tag. Then the His tag was split off by means of enterokinasedigestion. The purity of the protein was checked in the SDS gel (datanot shown).

For immunisation, rabbits were firstly initially immunised with theantigen and subsequently subjected to several booster immunisations.Finally, polyclonal antiserums were obtained from the blood of therabbits.

In order to check the neutralising effect of the polyclonal antiserums,firstly TcdB was pre-treated with polyclonal antiserum (dilution stage1:100) and incubated for 1 h at room temperature. Then the neutralisingeffect of the antiserums was checked as described in Example 2 with theaid of the CHO test. The measurement for the neutalising effect of theserums was how long the cells were protected from the cytotoxic effectof the TcdB.

The rabbits immunised with preparation A showed only a small antibodytiter (cf. Tab. 4), in addition the serum of these rabbits did not havea neutralising effect. This finding corresponds to the results known tothe specialist in the art of immunisation experiments with formalisedLCTs.

The animals which were immunised with preparation B did indeed develop adistinct titer (dilutable up to 1:1500), however this antiserum also didnot show any neutralising effect in the CHO test (Tab. 4).

The rabbits which had been immunised with the preparations C to Elikewise showed an antibody titer, and in addition their polyclonalserums showed a distinctly neutralising effect in the CHO test (Tab. 4).The polyclonal serum which was generated by immunisation withpreparation E showed the best neutralising characteristics here.

The serums which were produced by separate immunisation with thepreparations C and D showed a smaller neutralising effect, compared withthis.

The success of the immunisation with fragment D, which comprises a partof the inositol-binding region, proves that this section of the LCTs isan important region for the activation of the toxins. The binding of theIP6 in this toxin section leads to the activation of the autocatalyticprotease activity and hence also to the activation of the LCTs.

The neutralising effect of the region around the DSG motif proves thatantibodies which are directed against the active centre of the proteasecan inhibit the proteolytic activity. With such antibodies, therefore, aprotection from the toxic effects of the LCTs can also be achieved invivo.

With the immunisation with preparation E, only the two fragments of thesuccessful preparations C and D were used together. The success of theimmunisation with both TcdB fragments is based on the fact that in theanimals antibodies are induced which are directed towards the activecentre of the protease and also those antibodies which bind to theinositol phosphate-binding region. The effect of the antiserum istherefore based on the fact that for the first time specific antibodieswere able to be induced, which systematically prevent the activation ofthe toxin.

The autocatalytic cleavage of the toxins is important for the naturaluptake of the LTCs into their target cells, because only thus is theN-terminal fragment released, imparting the actual toxic activity, inthe target cells. The binding of specific antibodies in the environmentof the DSG motif of the aspartate protease and the inositol phosphatebinding site prevents the autocatalytic cleavage of the toxins. Inpatients, therefore, through the use of toxin fragments, which arenecessary for the autocatalytic cleavage of the LCTs, an effectiveimmunisation against LCTs can be achieved.

LITERATURE

-   Ausubel, F. M. et al.: “Current Protocols in Molecular Biology”    (2003), John Wiley and Sons. Inc.-   Rupnik et al. (2005) “Characterization of the cleavage site and    function of resulting cleavage fragments after limited proteolysis    of Clostridium difficile toxin B(TcdB) by host cells.” Mikrobiol    151, 199-208.-   Moos et al. (2000) “Purification and evaluation of large clostridial    cytotoxins that inhibit small GTPases of Rho and Ras subfamilies”    Meth Enzymol. 325: 114-125.-   Pfeifer et al. (2003) “Cellular Uptake of Clostridium difficile    toxins B” J. Biol. Chem. 278: 44535-41.-   Rao et al. (1998) “Molecular and biotechnological aspects of    microbial proteases” Microbiol. Mol. Biol. Rev. 62: 597-635.-   Salto et al. (1994) “In vitro characterization of nonpeptide    irreversible inhibitors of HIV proteases”, J. Biol. Chem. 269:    10691-8.-   Tang (1971) “Specific and irreversible inactivation of pepsin by    substrate-like Epoxides”, J. Biol. Chem. 246: 4510-17.

TABLE 1 Autocatalytic cleavage of TcdB₁₀₄₆₃ with the addition of definedinositol phosphates Concentration IP 1 mM 100 μM 10 μM 1, 5 − − − 1, 4 −− − 4, 5 − − − 1, 4, 5 − − − 2, 3, 5 − − − 1, 3, 5 − − − 1, 3, 5, 6 − −− 1, 2, 3, 4, 6 − − − 1, 3, 4 + − − 1, 3, 4, 5 + − − 3, 4, 6 + − − 1, 2,3, 4 + − − 1, 2, 3, 4, 5 + − − 1, 2, 3, 5, 6 + + − 1, 3, 4, 5, 6 + + −3, 4, 5, 6 + + − 2, 3, 4, 5, 6 + + − 1, 4, 5, 6 + + − 1, 2, 3, 4, 5,6 + + +

TABLE 2 Cell rounding (in %) of CHO cells after incubation withTcdB₁₀₄₆₃ - with or without EPNP pre-treatment Rounded cells [%] after 3h Dilution TcdB-10463 TcdB-10463 + EPNP 10⁻³ 100% 100% 10⁻⁴ 100% 100%10⁻⁵ 100% 50% 10⁻⁶ 100% 10% 10⁻⁷ 10% <5% 10⁻⁸ <5% <5%

TABLE 3 Comparison of the amino acid sequence AS 1653 to AS 1678 of thecatalytic centre of the protease function of TcdB with thecorresponding catalytic centres and protein regions of the toxinsTcdA, TcsL and Tcnα Toxin Homologous Sequence Range TcdB-104631653-QNMIVEPNYDLDDSGDISSTVINFSQ-1678 [SEQ ID NO: 1] TcdA-104631651-RNVVVEPIYNPDTGEDISTSL-DFSY-1675 [SEQ ID NO: 2] TcsL1654-QNLIVEPSYHLDDSGNISSIVINFSQ-1679 [SEQ ID NO: 3] Tcnα1641-CNVIVSGSNKLNSEGDLADT-IDVLD-1665 [SEQ ID NO: 4]

TABLE 4 Start of cell rounding (in h) of CHO cells after incubation withTcdB, which was pre-treated with polyclonal antiserum. PreparationAntibody Titer Start of cell rounding after A 1:100 1.5 h B  1:1500 3 hC 1:750 12-15 h D 1:500 9-12 h E  1:1000 >24 h

1. Method for preventing or relieving poisoning by LCT (=largeclostridial cytotoxins), comprising: administering to a host in needthereof at least one inhibitor or activator of an autocatalytic proteaseactivity of said LCT in an amount effective to prevent or relievepoisoning by LCT by stopping a cytotoxically effective fragment of saidLCT which has glycosyl transferase function to arrive in a host's cellcytosol.
 2. The method according to claim 1, wherein said at least oneinhibitor is an inhibitor of the autocataytic protease activity of oneor more of the following proteases: protease of Clostridium difficiletoxin A (TcdA), protease of Clostridium difficile toxin B (TcdB),protease of Clostridium sordellii lethal toxin (TcsL) or protease ofClostridium novyi α-toxin (Tcnα).
 3. The method according to claim 2,wherein the inhibitor is 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). 4.The method according to claim 1, wherein the activator is an inositolphosphate, preferably inositol hexaphosphate (IP6).
 5. The methodaccording to claim 2, wherein the inhibitor is a competitivelyinhibiting structural analogue of inositol phosphate.
 6. The methodaccording to claim 1, wherein the activator is a substance whichanalogous to IP6 promotes (autocatalytic) protease activity of LCTs. 7.The method according to claim 2, wherein the inhibitor is a chemicalsubstance that reduces an inositol phosphate concentration in gut lumenof mammals including humans.
 8. The method according to claim 2, whereinthe inhibitor is a chemical substance that reduces an inositol phosphateconcentration in mammalian cells.
 9. The method according to claim 2,wherein the inhibitor is a chemical substance, in particular a protein,more particularly an antibody and interacts with an active centre of theprotease in TcdB.
 10. The method according to claim 9, wherein theinhibitor interacts with SEQ ID NO:
 1. 11. The method according to claim2, wherein the inhibitor is a chemical substance, in particular aprotein, more particularly an antibody, which interacts with the DXGmotif at amino acid position AS 1665 of the TcdB protein according toSEQ ID NO: 1 and table 3 line
 1. 12. The method according to claim 2,wherein the inhibitor is a chemical substance, in particular a protein,more particularly an antibody which interacts with SEQ ID NO:
 2. 13. Themethod according to claim 2, wherein the inhibitor is a chemicalsubstance, in particular a protein, more particularly an antibody whichinteracts with a DXG motif at amino acid position AS 1662 of SEQ ID NO:2 and table 3 line
 2. 14. The method according to claim 2, wherein theinhibitor is a chemical substance, in particular a protein, moreparticularly an antibody which interacts with SEQ ID NO:
 3. 15. Themethod according to claim 2, wherein the inhibitor is a chemicalsubstance, in particular a protein, more particularly an antibody whichinteracts with a DXG motif at amino acid position AS 1666 of a TcsLprotein according to SEQ ID NO: 3 and table 3 line
 3. 16. The methodaccording to claim 2, wherein the inhibitor is a chemical substance, inparticular a protein, more particularly—an antibody which interacts withSEQ ID NO:
 4. 17. The method according to claim 2, wherein the inhibitoris a chemical substance, in particular a protein, more particularly anantibody and interacts with a TcdB protein region of AS 1400 to AS 2300according to TcdB amino acid sequence No. P18177 (SwissProt/TrEMBL) orwith protein regions of TcdA or TcsL or Tcnαhomologous thereto.
 18. Themethod according to claim 2, wherein the inhibitor is a chemicalsubstance, in particular a protein, more particularly an antibody andinteracts with a TcdB protein region of AS 1517 to AS 2142 according toTcdB amino acid sequence No. P18177 (SwissProt/TrEMBL) or with proteinregions of TcdA or TcsL or Tcnαhomologous thereto.
 19. The methodaccording to claim 2, wherein the inhibitor is a chemical substance, inparticular a protein, more particularly an antibody and interacts with aTcdB protein region of AS 1517 to AS 1593 or of AS 1918 to AS 2142according to TcdB amino acid sequence No. P18177 (SwissProt/TrEMBL) orwith protein regions of TcdA or TcsL or Tcnα homologous thereto. 20.Method for preventing or relieving poisoning by LCTcomprising:administering a medicament that (1.) is suited for administration as avaccine and (2.) as an antigen active ingredient (a) contains a TcdBprotein fragment which comprises at least a DXG motif at position 1665,preferably an amino acid sequence of amino acid positions AS 1653 to AS1678 [SEQ ID NO 1], and/or (b) contains a TcdA protein fragment whichcomprises at least a DXG motif at position 1662, preferably an aminoacid sequence of amino acid positions AS 1651 to AS 1675 [SEQ ID NO: 2],and/or (c) contains a TcsL protein fragment which comprises at least aDXG motif at position 1666, preferably an amino acid sequence of aminoacid positions AS 1654 to AS 1679 [SEQ ID NO: 3], and/or (d) contains aTcnα protein fragment which comprises at least an amino acid sequence ofAS 1641 to AS 1665 [SEQ ID NO: 4].
 21. The method according to claim 1,wherein said at least one activator of a protease activity is anactivator of the autocataytic protease activity of one or more of thefollowing proteases: protease of Clostridium difficile toxin A (TcdA),protease of Clostridium difficile toxin B (TcdB), protease ofClostridium sordellii lethal toxin (TcsL) or protease of Clostridiumnovyi α-toxin (Tcnα).
 22. The method according to claim 21, wherein theactivator interacts with one or more of the protease of Clostridiumdifficile toxin A (TcdA), Clostridium difficile toxin B (TcdB),Clostridium sordellii lethal toxin (TcsL) or Clostridium novyi α-toxin(Tcnα), initiates said protease activity and wherein a cytotoxicallyeffective fragment of said Clostridium difficile toxin A (TcdA),Clostridium difficile toxin B (TcdB), Clostridium sordellii lethal toxin(TcsL) or Clostridium novyi α-toxin (Tcnα), respectively, isautocatalytically split off via said protease activity outside a hostcell.
 23. The method of claim 1, wherein said activator activates saidprotease activity before the LCT has bound to the host cell and splitsoff the cytotoxically effective fragment of said LCT outside the hostcell.
 24. The method of claim 1, wherein said inhibitor inhibits saidprotease activity of said LCT by interacting with the LCT and preventingan autocataytic splitting off or autocleavage of the cytotoxicallyeffective fragment of said LCT via said protease activity.
 25. Methodfor stopping protease cleavage of a LCT, comprising providing aninhibitor of the autocataytic protease activity of one or more of thefollowing proteases: protease of Clostridium difficile toxin A (TcdA);protease of Clostridium difficile toxin B (TcdB); protease ofClostridium sordellii lethal toxin (TcsL) or protease of Clostridiumnovyi α-toxin (Tcnα), wherein the inhibitor interacts with the proteaseof Clostridium difficile toxin A (TcdA); Clostridium difficile toxin B(TcdB); Clostridium sordellii lethal toxin (TcsL) or Clostridium novyiα-toxin (Tcnα) respectively, and inhibits said protease activity toautocatalytically split off the cytotoxically effective fragment of saidClostridium difficile toxin A (TcdA); Clostridium difficile toxin B(TcdB); Clostridium sordellii lethal toxin (TcsL) or Clostridium novyiα-toxin (Tcnα), respectively, outside a host cell.